Active opioid peptides like other neuropeptides and polypeptide hormones are formed through the action of intracellular processing enzymes which cleave and then modify precursor proteins. The long-term goal of these studies is to better understand the enzymatic mechanisms responsible for the proteolytic processing of proenkephalin to bioactive opioids, with emphasis on the role of the new subtilisin-like processing enzymes (PC1 and PC2) which have recently been described in neuroendocrine tissues. Since proteolytic cleavage represents the first step of the enkephalin biosynthetic pathway, it is likely that regulatory mechanisms which control opioid peptide production may involve these crucial proteolytic enzymes. This proposal represents our continued efforts to describe the proteolytic processing of proenkephalin by PC1 and PC2. Co-transfection experiments of enzyme cDNAs with proenkephalin cDNA will help us to better understand the interaction of opioid precursors with processing enzymes in a cellular context, while our overexpression experiments, which have provided us with purified recombinant proenkephalin, PC1 and proPC2, will provide information on the regulation of enzyme activity and the identity of in vitro products of proenkephalin digested with PCs. Anti sense blockade will be used to investigate a regulatory role for PC2 in the generation of active opioids. Site-directed mutagenesis will be employed to study catalytically important domains within PC1; this technique will also be used to investigate the biochemical basis for ordered processing of proenkephalin. Taken together, these experiments should provide significant progress toward our goal of understanding regulatory mechanisms in opioid peptide biosynthesis. Deficiencies in the biosynthetic capacity for opioid peptides may be responsible for the addictive properties of opiate drugs in certain individuals; thus the study of enzymatic mechanisms regulating endogenous opioid production is of extreme importance. A thorough understanding of regulatory mechanisms in opioid peptide synthesis might one day lead to enzyme-based drugs serving as therapeutic agents in opiate addiction.